This invention relates to a DNA segment or vector system useful for sequencing long deoxyribonucleic acid (DNA) molecules and, more particularly, to a novel prokaryotic transposon.
It is known that certain complex DNA segments, known as transposons, are able to insert into many sites in the genome of their host organisms. That is, certain large segments of DNA called insertion sequences (ISs) when present in closely spaced pairs can move as a unit, carrying along the genes lying between them. These complex units constitute the transposon. They exist in prokaryotes, such as bacteria, as well as in eukaryotes.
Recently, a useful bacterial transposon referred to a Tn5 was discovered and characterized. It is a discrete 5.8 kilobase (kb) segment of bacterial DNA which can insert at high frequency into numerous sites in the chromosomes, plasmids, and temperate phages of gram negative bacteria. It encodes resistance to the aminoglycoside antibiotics kanamycin and neomycin in bacteria, and G418 (genticin) in eukaryotic cells. The restriction enzyme map of Tn5 is illustrated by Berg et al., Genetics 105, 813-828, (1983). Further background information on Tn5 can be had by reference to the recent review articles by Berg and Berg, Bio/Technology 1, 417-435 (1983); and Berg and Berg in Neidhardt et al., (eds.), "Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, " ASM, Washington, D.C., Ch. 63, pp. 1,071-1,109 (1987).
Two widely used methods for DNA sequence analysis are the base-specific chemical cleavage method [Maxam and Gilbert, Methods Enzymol. 65, 499-560 (1980)] and the enzymatic chain termination method [Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5,463-5,467 (1977)]. The chemical cleavage method usually requires knowledge of at least part of the restriction map of the DNA segment to be sequenced, it involves the handling of toxic chemicals, and is relatively time-consuming. The enzymatic chain termination method by comparison is easier, and for many applications has become the method of choice. Generally in this method the DNA segment of interest is cloned in an appropriate vector, and a short oligonucleotide complementary to sequences adjacent to the cloning site is used to prime DNA synthesis in the presence of base-specific DNA chain terminators. Typically, only a few hundred base pairs (bp) can be sequenced from the primer site, and thus a number of strategies have been devised to bring more distant regions near to the primer site. These include subcloning of small DNA fragments, isolation of nested sets of deletion derivatives either in vivo [Ahmed, Gene 39, 305-309 (1985)] or in vitro [Sanger et al., J. Mol. Biol. 143, 161-178 (1980); Barnes et al., Methods Enzymol. 65, 98-122 (1980); Deininger, Anal. Biochem. 135, 247-263 (1983); and Messing, Methods Enzymol. 101, 20-77 (1983)]; or making new oligonucleotide primers complementary to the end of each sequenced segment [Winnoto et al., Nature 324, 679-681 (1986)].
Because the foregoing sequencing methods are laborious, an improved method and vector for the sequencing of long DNAs would have significant use in molecular genetic analysis.